Genome-wide characterization of the CPA gene family in potato and a preliminary functional analysis of its role in NaCl tolerance

Background The cation/proton antiporter (CPA) superfamily plays a crucial role in regulating ion homeostasis and pH in plant cells, contributing to stress resistance. However, in potato (Solanum tuberosum L.), systematic identification and analysis of CPA genes are lacking. Results A total of 33 StCPA members were identified and classified into StNHX (n = 7), StKEA (n = 6), and StCHX (n = 20) subfamilies. StCHX owned the highest number of conserved motifs, followed by StKEA and StNHX. The StNHX and StKEA subfamilies owned more exons than StCHX. NaCl stress induced the differentially expression of 19 genes in roots or leaves, among which StCHX14 and StCHX16 were specifically induced in leaves, while StCHX2 and StCHX19 were specifically expressed in the roots. A total of 11 strongly responded genes were further verified by qPCR. Six CPA family members, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19, were proved to transport Na+ through yeast complementation experiments. Conclusions This study provides comprehensive insights into StCPAs and their response to NaCl stress, facilitating further functional characterization. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-024-10000-2.


Background
Salt stress severely impacts plant growth and yield, affecting over 800 million hectares of land globally and hindering sustainable agriculture [1,2].NaCl, the most soluble and widely distributed soil salt, is absorbed by roots, accumulating in tissues and resulting in damage to plants [1,3].Throughout evolution, plants developed various mechanisms to regulate the intracellular ion balance and resist salt stress, relying on ion transporters in cell and organelle membranes [4].
Potato (Solanum tuberosum L.) is the world's thirdlargest food crop, valued for its rich nutrition, high yield, and adaptability.However, potato is relatively sensitive to salt stress.Excess salt in the soil reduces photosynthetic rates, disrupts ion balance, and impairs osmotic regulation, affecting tuber growth and development, leading to yield loss. Identifying genes conferring resistance to abiotic stress is crucial for molecular breeding in potato.Although some CPA genes have been linked to NaCl stress response in other plants, detailed information on CPA in potatoes remains limited.Our study aimed to systematically identify the CPA superfamily in the potato genome and analyze their phylogenetic relationship, gene structure, and conserved motifs.Additionally, we investigated the expression patterns of StCPAs under salt (NaCl) treatment and confirmed the sodium ion transport function of StNHX1, StNHX2, StNHX3, StNHX5, StNHX6 and StCHX19.These findings lay the groundwork for further understanding of the role of StCPAs in NaCl stress responses.

Identification and characterization of CPAs in potato
To identify CPA members in potato, we performed a BLAST search against the potato protein database using known CPA sequences from Arabidopsis.We identified 33 StCPA members in the potato genome, categorized into three subfamilies: StNHXs (n = 7), StCHXs (n = 20), and StKEAs (n = 6).The genes were named StNHX1-StNHX7, StCHX1-StCHX20, and StKEA1-StKEA6 based on homologies with other species.Detailed information, such as gene name, coding region length, protein length, molecular weight, theoretical isoelectric point (pI), and subcellular localization predictions for all members, were analyzed (Table 1).Notably, all StCPAs contained a Na + / H + exchange domain, with lengths of 281 to 1,828 amino acid and molecular weights of 30 to 206 kDa.The theoretical pI ranged from 4.48 to 9.76, indicating different protein charges.The subcellular localization of StCPA predicted by WoLF PSORT, and most of them were on the plasma membrane, in line with the function of maintaining Na + homeostasis as transporters.In addition, StCPA are located on several organelles, including the endoplasmic reticulum, vacuole, cytoplasm, golgi apparatus and peroxisome.

Phylogenetic tree and chromosome localization analysis
To analyze the phylogenetic relationships of all StC-PAs members, we extracted full-length CPA protein sequences from potato, Arabidopsis, tomato, radish and grape, then aligned them to construct the neighbor-joining (NJ) phylogenetic tree (Fig. 1).
In total, 174 CPA proteins from five species (33 from potato, 40 from Arabidopsis, 26 from tomato, 48 from radish and 27 from grape) were categorized into three subfamilies: NHX, KEA, and CHX.Among them, the NHX group has 23 members, and the KEA group has 28 members.The CHX group is the most abundant subfamily, with 123 members, including 19, 22, 35, 27 and 20 from tomato, grape, radishe, Arabidopsis and potatoe, respectively.The phylogenetic tree indicated that the CPA gene family was highly conserved in different species.
The StCPAs were mapped on the 12 chromosomes, and they unevenly distributed across different chromosomes, with one to seven genes on each chromosome (Fig. 2).Chromosome 8 had the most CPA members (seven), followed by five on chromosome 6 and four on chromosome 1.Chromosomes 4 and 5 each had three members, while chromosomes 2, 3, 9, and 12 each had two members.The remaining chromosomes each contained one member.

Conserved motifs and gene structure analysis
To distinguish the differences among the three StCPA families, we conducted an analysis of the gene composition and structure of the StCPA proteins (Fig. 3).StCHX members exhibited three to eleven conserved motifs, while KEA members had four to five conserved motifs.In the StNHXs family, StNHX3 and StNHX6 had no conserved motifs, StNHX1 and StNHX2 had only one conserved motif, StNHX4, and StNHX5 had two conserved motifs, and StNHX7 had four conserved motifs (Fig. 3A).Notably, most StCPA members within the same subfamily had similar motif compositions, implying conserved functions among these proteins.Additionally, the number of motifs varied across different subfamilies, with CHXs being the most abundant, followed by KEAs and NHXs (Fig. 3B).This suggests that the functions of different subfamilies may have evolved different functions over time.
We further analyzed the structural diversity of these StCPA (Fig. 4).The results revealed significant variations in sequence length and the number of introns/ exons among StCPA members.The CHX family members had shorter sequence lengths and fewer exons, whereas the NHX and KEA family members had longer  [17].

RNA-seq analysis of the CPA gene superfamily under NaCl stress
To investigate the potential function of the StCPA superfamily under NaCl stress, we analyzed their expression patterns in leaves and roots under 200 mM NaCl treatment using RNA-seq data.The results revealed that the expression levels of 19 StCPAs were enhanced in roots or leaves under NaCl treatment (Fig. 5A).Notably, StCHX14 and StCHX16 exhibited specific high expression in leaves, while StCHX2 and StCHX19 are specifically expressed in roots, indicating that they may be specifically involved in regulating ion homeostasis in leaves or roots, thereby affecting NaCl tolerance (Fig. 5A and B).Interestingly, the remaining members (n = 14) of StCHX were not induced

Quantitative expression analysis of StCPAs under NaCl stress
The expression of this family was further validated by qPCR analysis under NaCl stress (Fig. 6).We selected

Effect of NaCl stress on yeast cell growth
In order to verify whether these responsive genes own the Na + transport function, we selected seven genes, which strongly responded to NaCl stress both in roots and leaves, for validation using defective yeast expression experiments.These genes included StNHX1, StNHX2, StNHX3,  the AXT3K strain (Fig. 7).These results are consistent with previous research [15,37].
To date, numerous studies have reported the involvement of the CPA gene superfamily in NaCl tolerance in plants [9,38].Our qPCR results demonstrated that all 11 selected StCPA genes were upregulated in response to NaCl stress in leaves or roots, indicating that upregulating the expression of antiporters is an important mechanism for coping with NaCl stress.SOS1 (Salt overly sensitive 1) protein, a Na + /H + antiporter found in various plants, is induced by NaCl stress and functions in Na + efflux to enhance NaCl tolerance in plants [1,3,39].In potato, StNHX1 is the homologous gene of AtSOS1.We observed significant induction of StNHX1 by NaCl stress in both leaves and roots of potato, consistent with our expectations.Furthermore, we noticed tissue-specific differences in gene expression.RNA-Seq showed that StCHX14 and StCHX16 were specifically expressed in leaves, while StCHX2 and StCHX19 were specifically expressed in roots.qPCR data showed that the expression level of StNHX4 increased by 7 folds in the roots after 24 h of NaCl stress, while it was not significant in the leaves.This further proved that StNHX4 play the major role in the roots.StKEA4 showed a significant increase in expression levels, but not induced in the roots, suggesting that this gene mainly plays a role in the leaves.So, it is speculated that different members have different tissues-specific functional divergence of regulating NaCl tolerance.
In addition, we observed that the expression levels of all StNHX and most (5/6) StKEA members significantly increased in roots or leaves under NaCl stress.
For StCHX subfamily, six members (StCHX9, StCHX15, StCHX2, StCHX19, StCHX14, and StCHX16) induced by NaCl stress, while most of (n = 14) members were not sensitive to NaCl stress.This indicated StNHX and StKEA play major roles in coping with NaCl stress, while StCHX subfamily members may be induced by various forms of salt stress.
The yeast system for rapid verification of CPA function has been widely employed in various plants [15,40,41].For instance, heterologous expression of ZmNHX5, ZmCHX2, ZmCHX3, ZmCHX5, ZmCHX17, and ZmKEA2 restored the Na + resistance of the yeast mutant AXT3K, and the NaCl tolerance function of ZmCHX2 and ZmNHX5 was confirmed in transgenic plants [40].Similarly, the wheat CPA gene superfamily TaNHX4 enhanced the survival rate of Escherichia coli under various abiotic stresses [15].In Arabidopsis, the AtKEA subfamily, except for AtKEA3, improved the tolerance of yeast mutant strains to high K + stress [41].To verified which member own ion transport function, a total of seven genes were applied to a yeast system.The results showed that heterologous expression

Conclusions
In this study, we conducted a comprehensive investigation of the CPA superfamily members in potato.A total of 33 StCPA genes were identified and categorized into three subfamilies: NHX, KEA, and CHX, with significant differences in conserved motifs and exons.More than half of the StCPA genes were induced by NaCl stress, exhibiting varying magnitudes and response times.Notably, StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, and StCHX19 were found to transport Na + and enhance NaCl tolerance in defective yeast mutants.These findings provide valuable insights for future research on the biological functions and molecular mechanisms of these potato CPA genes in response to NaCl stress.

Planting and CPA gene superfamily RNA-Seq analysis
The potato genotype A056 was propagated using MS medium and cultured for 15 days in a growth room (conditions: light duration of 16 h, dark duration of 8 h; temperature of 21 ℃) and transplanted into a matrix block (the matrix block material is composed of coconut bran, peat, and wood, and after high-temperature sterilization, it was wrapped in a degradable mesh like non-woven fabric to form 41 mm × 42 mm sized matrix block).The seedlings were cultured for 2-3 days and grown normally over the course of 15 days.Add NaCl into tap water and prepare 200 mM NaCl aqueous solution and pour it into a seedling tray for potato seedlings to absorb and form NaCl stress.Leaf and root samples were collected from seedlings at 0 h, 6 h, 12 h, 24 h, and 48 h after NaCl treatment and flash-frozen in liquid nitrogen.Three biological replicates for each treatment were conducted.RNA was extracted using the TRIzol reagent method, and StCPA transcriptome analysis was performed.RNA-Seq was completed by the Beijing Annuo Company, China.The FPKM expression value was determined from the sequencing data and were further screened by heatmap drawing using TBtools software.

Quantitative real-time PCR
The RNA samples for qPCR were identical to those used for RNA-Seq analysis.The first strand cDNA synthesis from total RNA reverse transcription was performed using the PrimeScript ™ RT reagent Kit with gDNA Eraser (Perfect Real Time) from TaKaRa.Quantitative RT-PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Code No. RR820A/B) from TaKaRa on a StepOnePlus Real-Time PCR Instrument (Applied Biosystems).Tubulin was employed as an internal control to normalize the samples.Primer design was conducted using Primer Premier 5 software and is outlined in Supplementary Table S1.All experiments were conducted with biological triplicates.

Cloning of gene
Plant material accession A056 was utilized for total RNA extraction and reverse transcription polymerase chain reaction to generate cDNA sequences (an identical method to the described qPCR procedure).Primers were designed based on the CDS of StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 from potato DM genomes.Detailed primer sequences can be found in Supplementary Table S2.In order to clone the coding region sequences of the StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 genes, the cDNA of the A056 material was employed as a template for PCR amplification using the Supplementary Table S2 primers.The sequences of the StNHX1, StNHX2, StNHX3, StNHX5, StNHX6, StNHX7, and StCHX19 genes were amplified and inserted into the P416 vector using the seamless cloning method (In-Fusion ® Snap Assembly Master Mix) (TaKaRa).Finally, vector construction was verified by sequencing.The constructed vectors included P416-StNHX1, P416-StNHX2, P416-StNHX3, P416-StNHX5, P416-StNHX6, P416-StNHX7, and P416-StCHX19.

Fig. 1
Fig. 1 Phylogenetic tree of CPA proteins in potato, Arabidopsis, tomato, radish and grape.Neighbor-joining tree constructed using MEGA 11, depicting the phylogenetic relationship among 174 CPA proteins, comprising 40 AtCPAs, 33 StCPAs, 26 SlCPAs, 48 RsCPAs and 27 VvCPAs.The NHX, KEA, and CHX subfamilies encompass 23 members, 28 members, and 123 members, respectively the 6th hours (> 2-fold and > 1.5-fold) and remained this level; StNHX1, StNHX5, StNHX6, StCHX19, and StKEA4 showed a trend of upregulation and then downregulation.Among them, StNHX1, StNHX5, StNHX6, and StCHX19 showed the highest upregulation at the 12th hour and downregulation at the 24th hour, while StKEA4 showed the highest upregulation at the 6th hours and gradually downregulated thereafter.The expression level of StNHX2 was steadily upregulated in the leaves, while the other genes StNHX4, StKEA1, and StKEA2 were not induced by NaCl stress in the leaves.In addition, we noticed that the induced expression levels of StNHX3, StNHX4, StCHX19, StKEA1, and StKEA2 were higher in the roots than in the leaves, while the induced expression levels of StNHX1, StNHX5, StNHX6, and StKEA4 in the leaves were higher in the roots.In summary, different StCPA members exhibited various responsive patterns, indicating their diverse functions in response to NaCl stress.

Fig. 2
Fig. 2 Chromosomal position of StCPA genes.Distribution of StCPA genes in potato chromosomes.Chromosome numbers are shown within each bar.The scale on the left is in megabases (Mb)

Fig. 3
Fig. 3 Conserved motifs of StCPA proteins.A Analysis of the conserved domains of the StCPA proteins.The 10 motifs are depicted using different color ranges.B Number of conserved motifs in StNHX, StKEA, and StCHX subfamilies

Fig. 4
Fig. 4 Structural analysis of the StCPA gene superfamily.A Structures of StCPA gene superfamily.Introns and exons are represented as thin lines and green boxes, respectively.B Number of exons in StNHX, StKEA, and StCHX subfamilies

Table 1
Physiochemical properties of StCPA genes